Project Summary Diabetic retinopathy and Retinal Vein Occlusion (RVO) account for most new cases of blindness in the United States. These diseases are marked by retinal swelling (edema) and new leaky vessel formation (neovascularization) triggered by pathologically high concentrations of Vascular Endothelial Growth Factor (VEGF). The primary treatment is VEGF-blocking drugs to control the neovascularization and edema, but neuronal damage from oxygen starvation continues to progress. Steroids treat edema (swelling) but do not address neovascularization and they have adverse side effects. Some VEGF is needed for neuronal and vascular endothelial cell health, so alternative treatments are needed to regenerate and repair the retinal vasculature without blocking normal VEGF-signaling. We propose to use a recombinant protein mimetic of human Norrin, called Noregen?, as a safe regenerative therapy based on our patented technology. There is an excellent scientific basis for this proposed regenerative therapy. Norrin is a normal stimulator of retinal endothelial cells and is essential for making the retinal vasculature. Norrin also supports the repair and regrowth of blood vessels into retinal areas that have lost vessels and improves the organization and speed of vessel regrowth in a mouse model of oxygen-induced retinopathy. Additionally, our current results demonstrate that Norrin can counter VEGF-induced changes that increase the leakiness through endothelial cells caused by Plasmalemma Vesicle-Associated Protein (PLVAP). While elevated VEGF increases the expression of the PLVAP gene, we have found that Norrin reduces PLVAP gene expression. In Phase I we will develop a Noregen production process and conduct preclinical testing in animal models and with vascular endothelial cells from human donor retinas. Successful completion of Phase I will provide the basic process for making the Noregen protein and the test methods needed to confirm that the protein is also effective for stimulating endothelial cell replacement and the formation of a vascular endothelial cell barrier. In Aim 1, we will establish bacterial production and purification of Noregen protein, with Frizzled-4 binding activity. Recombinant Noregen will be synthesized in bacteria, refolded into a biologically active state and purified using cation-exchange chromatography. In Aim 2, we will test Noregen protein?s safety using a rat intraocular injection model. Purification steps will be added as required based on the results to reduce bacterial endotoxin. In Aim 3, we will confirm Noregen?s efficacy to support regeneration of the retinal vasculature in a mouse model and maintenance of the endothelial barrier in cultured primary Human Retinal Endothelial Cells. Completion of Phase I will permit Retinal Solutions to submit a Phase II application where Noregen production will be transferred into a current Good Manufacturing Process (cGMP) facility, and the therapeutic grade product used for additional pre-clinical safety testing to meet the Food and Drug Administration requirements for intraocular drugs.